1. Field of the Invention
The present invention relates generally to the field of molecular biology and medicine. More particularly, it concerns methods of generating dopaminergic neurons from pluripotent cells.
2. Description of Related Art
Cell populations that retain the ability to differentiate into numerous specialized cell types are useful for developing large numbers of lineage specific differentiated cell populations. These lineage specific differentiated cell populations are contemplated to find use in cell replacement therapies for patients with diseases resulting in loss of function of a defined cell population. In addition to their direct therapeutic value, lineage specific differentiated cells are also valuable research tools for a variety of purposes including in vitro screening assays to identify, confirm, and test for specification of function or for testing delivery of therapeutic molecules to treat cell lineage specific disease.
In the case of Parkinson's disease, for example, it is the loss of midbrain dopaminergic (DA) neurons that results in the appearance of disease symptoms. Thus, there is need for methods of producing DA neuronal cells from pluripotent cells, since such cells could be used both therapeutically and in disease models, e.g., to identify new therapeutics for treatments for Parkinson's disease.
Various efforts have been made to generate midbrain DA neurons from pluripotent cells. For example, methodologies for generating midbrain DA neurons from pluripotent cells typically require use of both LDN-193189, an inhibitor of BMP signaling (inhibits ALK 1/2/3/6, blocks SMAD 1/5/8), and SB-431542, an inhibitor of TGF-beta signaling (inhibits ALK 4/5/7, blocks SMAD 2/3), as described, e.g., in WO2013/067362. Since these methods utilize the combination of two inhibitors of Small Mothers Against Decapetaplegic (SMAD) signaling, these methods are typically referred to as “dual SMAD inhibition”, or “dual SMADi.” Although it may be desirable to create a method for generation of midbrain dopaminergic (mDA) neurons from induced pluripotent stem (iPS) cells using only a single SMAD inhibitor, such efforts have thus far been a failure. Clearly, there exists a need for methods of generation of mDA neurons from iPS cells using only a single SMAD inhibitor.